Expansion of haemopoietic precursors

ABSTRACT

The present invention relates to a method of transplanting haematopoietic precursor cells into a subject in need thereof which involves culturing the haematopoietic precursor cells in the presence of a population of cells enriched for STRO-1 bright  cells. The method of the present invention is useful in the treatment of haematological disorders.

This application is a §371 national stage of PCT InternationalApplication No. PCT/AU2009/001145, filed Sep. 3, 2009, and claims thebenefit of U.S. Provisional Application No. 61/190,967, filed Sep. 3,2008, which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates the present invention relates to a methodof transplanting haematopoietic precursor cells into a subject in needthereof which involves culturing the haematopoietic precursor cells inthe presence of a population of cells enriched for STRO-1^(bright)cells. The method of the present invention is useful in the treatment ofhaematological disorders.

BACKGROUND OF THE INVENTION

For more than a decade, umbilical cord blood (CB) has been investigatedclinically as an alternative source of hematopoietic progenitors forallogeneic transplantation of patients lacking an HLA-matched marrowdonor. Fewer T-cells and/or less developed T-cells in CB compared tomarrow allows for the possibility that CB grafts will produce less Graftvs. Host Disease (GVHD), the major cause of morbidity and mortality inthe allogeneic transplant setting. Other potential advantages includethe ability to markedly increase the number of allografts available andthus the number of patients who could be transplanted, given theavailability and ease of collecting CB from placental veins prior todisposal of the placenta, compared to collecting bone marrow orperipheral blood progenitor cells (PBPCs) from living donors. This newsource of hematopoietic progenitors has allowed CB banks to targetcollection of units with human leukocyte antigen (HLA) types such asthose of minority African American and Hispanic populations, which areunder-represented in the National Marrow Donor Program Registry.

Since the first CB transplant performed in 1988, more than 5,000patients world-wide have received related or unrelated CB transplantsfor a variety of malignant and non-malignant diseases. The majority ofCB recipients have been children although adults are increasinglyreceiving CB transplants when an HLA-matched donor is not available. Theprogression-free survival rates reported thus far are comparable toresults achieved following allogeneic bone marrow transplantation(Barker J N et al., (2001)). Moreover, there are many reports of whatappears to be less GVHD than that associated with bone marrowtransplants, despite the use of CB grafts with substantially moredonor-recipient HLA disparity than that tolerated in recipients ofmarrow or PBPC allografts. The major disadvantage of CB is the low celldose, which results in slower time to engraftment and higher rates ofengraftment failure when compared to bone marrow transplantation (KeenanN A et al. (1993)). In studies of CB transplantation published byKurtzberg (Kurtzberg J., (1996)), Gluckman (Gluckman et al., (1997)),Rubinstein (Rubinstein P., (1998), Rizzieri (Rizzieri D A et al.,(2001)), and Laughlin (Laughlin M J et al., (2001)) the median times toan absolute neutrophil count (ANC) of ≧0.5×10⁹/L ranged from 22 to 34days. Median times to a transfusion-independent platelet count ≧20×10⁹/Lvaried from 56 to over 100 days, with engraftment failure rates of12-18%. However, the engraftment failure rate for the adult patients(>18 years old and/or >45 Kg) in those series was substantially higher,ranging from 10-62%. It is these larger, adult patients, who mightbenefit the most from the ex vivo expansion of CB progenitor cells.

From the studies referenced above, there appears to be a thresholdeffect in the total nucleated cell (TNC) dose of unmanipulated CBinfused and time to engraftment. In Gluckman's study, engraftment andsurvival were superior in patients who received ≧3.7×10⁷ TNC/Kg. Thislarge a cell dose is not generally available for patients weighing morethan 45 kg. For adult patients, it appears that recipients of ≧1.0×10⁷TNC/Kg had more favorable engraftment than recipients of lower celldoses. Kurtzberg et al., reported a linear correlation between thenumber of CB nucleated cells infused and time to neutrophil engraftment(p<0.002) in the unrelated CB transplant setting. These data suggestthat giving more CB cells may result in faster neutrophil engraftment.

SUMMARY OF THE INVENTION

The present inventors have developed a method for expanding haemopoieticprogenitor cells (HPCs) by co-culturing with a population of cellsenriched for STRO-1^(bright) cells or progeny thereof. The expanded HPCscan be used for transplantation into a subject in need thereof, such anindividual with a haematological disorder.

Accordingly, the present invention provides a method of transplantinghaematopoietic precursor cells into a subject in need thereof, themethod comprising:

-   -   culturing haematopoietic precursor cells in the presence of a        population of cells enriched for STRO-1^(bright) cells or        supernatant or progeny derived therefrom, wherein such        STRO-1^(bright) cells are mesenchymal precursor cells (MPC)        which comprise mesenchymal precursor cells capable of giving        rise to colony forming unit-fibroblasts (CFU-F), so as to expand        the haematopoietic precursor cells; and    -   administering the expanded haematopoietic precursor cells to a        subject.

In one embodiment of the invention the population of cells enriched forSTRO-1^(bright) cells are allogeneic cells.

In another embodiment, the STRO-1^(bright) enriched cells are grownto >70% confluency for cord blood co-culture by about four days afterinitiating culture.

In another embodiment, the ratio of STRO-1^(bright) enriched cells tohematopoietic precursor cells at the start of the co-culture is about1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about1:10.

The STRO-1^(bright) enriched cells may be derived from any suitabletissue source. Examples of suitable tissue sources include bone marrow,blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain,kidney, liver, heart, retina, brain, hair follicles, intestine, lung,lymph node, thymus, bone, ligament, tendon, skeletal muscle, dermis andperiosteum.

In another embodiment, the haematopoietic precursor cells are derivedfrom cord blood. The haematopoietic precursor cells may or may not beisolated from the cord blood prior to expansion. Thus, in one embodimentthe method comprises co-culturing unmanipulated cord blood cells withthe population of cells enriched for STRO-1^(bright) cells or progenythereof.

In another embodiment of the invention the expanded haematopoieticprecursor cells comprise CFU-GM cells. The expanded haematopoieticprecursor cells may comprise at least 1×10⁴ CFU-GM cells per kg ofsubject body weight.

In another embodiment of the invention, haematopoietic reconstitutionoccurs in the subject following administration of the expandedhaematopoietic precursor cells. For example, haematopoieticreconstitution may occur in the subject within 30 days, more preferablywithin 25 days, more preferably within 20 days, more preferably within15 day and more preferably within 10 days of administration of theexpanded haematopoietic precursor cells.

In yet another embodiment, haematopoietic reconstitution occurs in theabsence of an adverse immune response. In this embodiment,administration of the expanded haematopoietic precursor cells does notresult in significant graft rejection.

Haematopoietic reconstitution may determined by any one of a number ofsuitable measurements. For example, haematopoietic reconstitution may bedetermined by neutrophil engraftment, platelet engraftment, lymphoidengraftment, erythroid engraftment and/or megakaryocyte engraftment.

The method of the invention may further comprise administering to thesubject one or more factors which enhances differentiation of thehaematopoietic precursor cells to a specific haematopoietic lineagecell. The factor which enhances differentiation may be administeredsimultaneously with the expanded haematopoietic precursor cells, orseparately after administration of the expanded haematopoietic precursorcells.

The haematopoietic lineage cell resulting from the differentiation maybe, for example, a B-cell, T-cell, dendritic cell, monocyte, neutrophil,macrophage, natural killer cell, granulocyte, erythrocyte, eosinophil,megakaryocyte, platelet, bone marrow, splenic, dermal, or stromal cell.

The factor which enhances differentiation may be, for example, a stemcell factor (SCF), GM-SCF, M-CSF, G-CSF, MGDF, EPO, FLT3-ligand, IL-1,IL-2, IL-3, IL-4, IL-6, IL-7, IL-11, TNFα or thrombopoietin.

Transplantation of the expanded haematopoietic precursor cells may beeffected together with the co-cultured MPC or progeny thereof, and/ortogether with supernatant or one or more soluble factors derived fromthe co-cultured MPC or progeny thereof.

In one embodiment, administration of haematopoietic precursor cellsexpanded in accordance with the method of the present invention leads toreduced risk of graft versus host disease when compared toadministration of haematopoietic precursor cells that have not beensubject to ex vivo expansion. In another embodiment, administration ofhaematopoietic precursor cells expanded in accordance with the method ofthe present invention leads to reduced risk of graft versus host diseasewhen compared to administration of haematopoietic precursor cells thathave been expanded by methods other than those of the present invention.

It will be appreciated that the method of the invention may be used inthe treatment of a range of haematologic disorders.

For example, the method of the invention may be used in the treatment ofa disorder of platelet number and/or function such as thrombocytopenia,idiopathic thrombocytopenic purpure (ITP), or a disorder related toviral infection, drug abuse or malignancy.

In another example, the method of the invention may be used in thetreatment of a disorder of erythrocyte number and/or function, such asan anaemia. Examples of anaemias that may be treated include aplasticanaemia, autoimmune haemolytic anaemia, blood loss anaemia, Cooley'sanaemia, Diamond-Blackfan anaemia, Fanconi anaemia, folate (folic acid)deficiency anaemia, haemolytic anaemia, iron-deficiency anaemia,pernicious anaemia, sickle cell anaemia, thalassaemia or PolycythemiaVera.

In another example, the method of the invention may be used in thetreatment of a disorder of lymphocyte number and/or function, such as adisorder caused by a T-cell or B-cell deficiency. Examples of disordersof lymphocyte number and/or function are AIDS, leukemias, lymphomas,Hodgkins lumphoma, chronic infections such as military tuberculosis,viral infections, rheumatoid arthritis, systemic lupus erythematosus, orhereditary disorders such as agammaglobulinemia, DiGeorge anomaly,Wiskott-Aldrich syndrome, or ataxia-telangiectasia.

In another example, method of the invention may be used in the treatmentof a disorder of multilineage bone marrow failure, which may be theresult of radiotherapy or chemotherapy or malignant replacement. Forexample, the disorder may be a myelofibrosis, acute myelogenous leukemia(AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia(ALL), chromic myelogenous leukemia (CML), chronic lymphocytic leukemia(CLL)), Non-Hodgkin's lymphoma (NHL), Hodgkin's Disease (HD), multiplemyeloma (MM), or a secondary malignancy disseminated to bone.

The present invention is applicable to a wide range of animals. Forexample, the subject may be a mammal such as a human, dog, cat, horse,cow, or sheep. In one embodiment the subject is a human.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT OF THE INVENTION

The present invention provides method for the ex viva expansion ofumbilical cord blood derived HPCs by co-culture with mesenchymalprecursor cells (MPCs). Such expanded HPCs are useful in the treatmentof conditions such as haematopoietic malignancies and in allogeneic celltherapy promoting regeneration of bone marrow.

As used herein, the term “expanding” or “expansion” refers to a processof cellular proliferation. Cells that undergo expansion maintain theircell renewal properties.

The present invention therefore provides a method of transplantinghaematopoietic precursor cells into a subject in need thereof, themethod comprising:

-   -   culturing haematopoietic precursor cells in the presence of a        population of cells enriched for STRO-1^(bright) cells or        supernatant or progeny derived therefrom, wherein such        STRO-1^(bright) cells are mesenchymal precursor cells (MPC)        which comprise mesenchymal precursor cells capable of giving        rise to colony forming unit-fibroblasts (CFU-F), so as to expand        the haematopoietic precursor cells; and    -   administering the expanded haematopoietic precursor cells to a        subject.

The term “supernatant” refers to the non-cellular material comprisingone or more soluble factors produced following the ex viva culture ofmesenchymal precursor cells, and/or progeny cells thereof, in a suitablemedium, preferably a liquid medium. Typically, the supernatant isproduced by culturing the cells in the medium under suitable conditionsand time, followed by removing the cellular material by a process suchas centrifugation. The supernatant may or may not have been subjected tofurther purification steps before administration. In a preferredembodiment, the supernatant comprises less than 10⁵, more preferablyless than 10⁺, more preferably less than 10³ and even more preferably nolive cells.

The term “one or more soluble factors” refers to molecules, typicallyproteins, secreted by the MPCs and/or progeny cells thereof, duringculture.

In one embodiment of the invention the population of cells enriched forSTRO-1^(bright) cells are allogeneic cells. The allogeneic cells may beobtained from an individual with a close HLA match to the subject. Anadvantage of the present inventions is that the allogenic cells may beproduced commercially in hulk for “off-the-shelf” use in ex vivoexpansion of haematopoietic precursor cells.

An “off-the shelf” source offers major potential advantages over familymember-derived STRO-1^(bright) cells. First, the cells can be madeavailable for immediate use without the need for lengthy processing orthe possibility of contamination during culture. The development ofmaster cell banks from young, healthy volunteers offers a way to bypassdisease-related decline in stem cell function providing the optimalsource of STRO-1^(bright) cells for the cord blood co-cultures. Finally,standardization of selection and isolation procedures provides a veryreproducible product.

A further advantage of the method of the invention is that sufficientnumbers of STRO-1^(bright) enriched cells used for co-culture with cordblood cells can be obtained considerably faster than with previousmethods of de novo generation of MPCs from a bone marrow donor. Thisallows for patients who are in fragile remission prior to transplant tobe treated sooner and thus reduce the likelihood of relapse.

The haematopoietic precursor cells may be derived from any suitablesource, one of which is cord blood. It is not necessary to isolate thehaematopoietic precursor cells prior to expansion. Accordingly, themethod of the invention may involve co-culturing cord blood with apopulation of cells enriched for STRO-1^(bright) cells or supernatant orprogeny derived therefrom. An advantage of this embodiment is that itobviates the need for isolation of CD34⁺ or CD133⁺ cells from cord bloodprior to expansion, therefore minimising manipulation and loss ofhaematopoietic precursor cells.

Engraftment can be facilitated by co-administration of a differentiationfactor such as a stem cell factor (SCF), GM-SCF, M-CSF, G-CSF, MGDF,EPO, FLT3-ligand, IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-11, TNFα orthrombopoietin. Administration of the differentiation factor may occurat the time of administration of the HPCs and/or at regular intervalsafter administration of the HPCs.

The haematopoietic lineage cell resulting from the differentiation maybe, for example, a B-cell, T-cell, dendritic cell, monocyte, neutrophil,macrophage, natural killer cell, granulocyte, erythrocyte, eosinophil,megakaryocyte, platelet, bone marrow, splenic, dermal, or stromal cell.

Accordingly, the methods of the invention also extend to the optionaluse of one or more differentiation factors for facilitatinghaematopoietic reconstitution post infusion of expanded HPCs.

Cells Enriched for STRO-1^(bright) Cells

In one embodiment the method of the present invention involvesco-culturing of haematopoietic precursor cells with a population ofcells enriched for STRO-1^(bright) cells, wherein such STRO-1^(bright)cells are mesenchymal precursor cells (MPC) which comprise mesenchymalprecursor cells capable of giving rise to colony formingunit-fibroblasts (CFU-F).

MPCs are non-hematopoietic progenitor cells that are capable of forminglarge numbers of multipotential cell colonies. The enrichment of adultMPCs is described in detail in WO 01/04268, the entire contents of whichare incorporated by reference. The term “MPCs” according to the presentinvention is also understood to include the Multipotential Expanded MPCprogeny (MEMPs) as defined in WO 2006/032092.

Mesenchymal precursor cells (MPCs) are cells found in bone marrow,blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain,kidney, liver, heart, retina, brain, hair follicles, intestine, lung,lymph node, thymus, bone, ligament, tendon, skeletal muscle, dermis, andperiosteum; and are capable of differentiating into different germ linessuch as mesoderm, endoderm and ectoderm. Thus, MPCs are capable ofdifferentiating into a large number of cell types including, but notlimited to, adipose, osseous, cartilaginous, elastic, muscular, andfibrous connective tissues. The specific lineage-commitment anddifferentiation pathway which these cells enter depends upon variousinfluences from mechanical influences and/or endogenous bioactivefactors, such as growth factors, cytokines, and/or localmicroenvironmental conditions established by host tissues. Mesenchymalprecursor cells are non-hematopoietic progenitor cells which divide toyield daughter cells that are either stem cells or are precursor cellswhich in time will irreversibly differentiate to yield a phenotypiccell.

In one embodiment, the STRO-1⁺ cells used in the present invention arealso TNAP⁺, STRO-3⁺ (TNSAP), VCAM-1⁺, THY-1⁺, STRO-2⁺, CD45⁺, CD146⁺,3G5⁺ or any combination thereof. For example, the STRO-1^(bright) cellsmay additionally be one or more of VCAM-1⁺, THY-1⁺, STR-2⁺, STRO-3⁺(TNSAP) and/or CD 146⁺.

In one embodiment, the mesenchymal precursor cells are perivascularmesenchymal precursor cells as defined in WO 2004/85630.

A cell is “positive” for a given marker if it is either a low (lo ordim) or a high (bright, bri) expresser of that marker depending on thedegree to which the marker is present on the cell surface, where theterms relate to intensity of fluorescence or other colour used in thecolour sorting process of the cells. The distinction of lo (or dim ordull) and bri will be understood in the context of the marker used on aparticular cell population being sorted.

The term “bright”, when used herein, refers to a marker on a cellsurface that generates a relatively high signal when detectablylabelled. Whilst not wishing to be limited by theory, it is proposedthat “bright” cells express more of the target marker antigens. Forinstance, STRO-1^(bri) cells produce a greater fluorescent signal, whenlabelled with a FITC-conjugated STRO-1 antibody as determined by FACSanalysis, than non-bright cells (STRO-1^(dull/dim)). In another example,STRO-1^(bright) cells have 2 log magnitude higher expression of STRO-1surface expression relative to an isotype matched negative control. Bycomparison, STRO-1^(dim) and/or STRO-1^(intermediate) cells have lessthan 2 log magnitude higher expression of STRO-1 surface expression,typically about 1 log or less higher expression over the isotype matchednegative control.

The population of cells used in the present invention is preferablyenriched for STRO-1⁺ cells relative to STRO-1^(dim) and/orSTRO-1^(intermediate) cells.

When used herein the term “TNAP” is intended to encompass all isoformsof tissue non-specific alkaline phosphatase. For example, the termencompasses the liver isoform (LAP), the bone isoform (BAP) and thekidney isoform (KAP). In a preferred embodiment, the TNAP is BAP. In aparticularly preferred embodiment, TNAP as used herein refers to amolecule which can bind the STRO-3 antibody produced by the hybridomacell line deposited with ATCC on 19 Dec. 2005 under the provisions ofthe Budapest Treaty under deposit accession number PTA-7282.

It is preferred that a significant proportion of the MPCs are capable ofdifferentiation into at least two different germ lines. Non-limitingexamples of the lineages to which the multipotential cells may becommitted include bone precursor cells; hepatocyte progenitors, whichare multipotent for bile duct epithelial cells and hepatocytes; neuralrestricted cells, which can generate glial cell precursors that progressto oligodendrocytes and astrocytes; neuronal precursors that progress toneurons; precursors for cardiac muscle and cardiomyocytes,glucose-responsive insulin secreting pancreatic beta cell lines. Otherlineages include, but are not limited to, odontoblasts, dentin-producingcells and chondrocytes, and precursor cells of the following: retinalpigment epithelial cells, fibroblasts, skin cells such as keratinocytes,dendritic cells, hair follicle cells, renal duct epithelial cells,smooth and skeletal muscle cells, testicular progenitors, vascularendothelial cells, tendon, ligament, cartilage, adipocyte, fibroblast,marrow stroma, cardiac muscle, smooth muscle, skeletal muscle, pericyte,vascular, epithelial, glial, neuronal, astrocyte and oligodendrocytecells.

In another embodiment, the MPCs are not capable of giving rise, uponculturing, to hematopoietic cells.

The present invention also relates to use of supernatant or solublefactors obtained from MPCs and/or progeny cells thereof (the latter alsobeing referred to as expanded cells) which are produced from the invitro culture of freshly isolated MPCs. Expanded cells of the inventionmay a have a wide variety of phenotypes depending on the cultureconditions (including the number and/or type of stimulatory factors inthe culture medium), the number of passages and the like. In certainembodiments, the progeny cells are obtained after about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, or about 10passages from the parental population. However, the progeny cells may beobtained after any number of passages from the parental population.

The progeny cells may be obtained by culturing in any suitable medium.The term “medium”, as used in reference to a cell culture, includes thecomponents of the environment surrounding the cells. Preferably, themedium used in the co-culture methods of the present invention is aliquid medium.

Preferably, the culture medium is supplemented with one or more growthfactors or cytokines which support the expansion of HPCs. Preferably,the cytokines are early acting cytokines, such as, but not limited torecombinant metHu stem cell factor (SCF), flt-3 ligand (FLT3), IL-1,IL-2, IL-3, L-6, IL-10, IL-12, tumor necrosis factor-α andthrombopoietin.

Late acting cytokines can also be used. These include for example,granulocyte colony stimulating factor (G-CSF), granulocyte/macrophagecolony stimulating factor (GM-CSF), erythropoietin (EPO), LIF andmacrophage growth factor (M-CSF).

Multipotential Expanded MPC Progeny (MEMPs) are defined in WO2006/032092. Methods for preparing enriched populations of MPC fromwhich progeny may be derived are described in WO 01/04268 and WO2004/085630. In an in vitro context MPCs will rarely be present as anabsolutely pure preparation and will generally be present with othercells that are tissue specific committed cells (TSCCs). WO 01/04268refers to harvesting such cells from bone marrow at purity levels up toabout 90%. The population comprising MPC from which progeny are derivedmay be directly harvested from a tissue source, obtained from a mastercell bank, or alternatively it may be a population that has already beenexpanded ex vivo.

For example, the progeny may be obtained from a harvested, unexpanded,population of substantially purified MPC, comprising at least about 0.1,1, 5, 10, 20, 30, 40, 50, 60, 70, 80 or 95% of total cells of thepopulation in which they are present. This level may be achieved, forexample, by selecting for cells that are positive for at least onemarker selected from the group consisting of TNAP, STRO-1^(bright),3G5⁺, VCAM-1, THY-1, CD146 and STRO-2.

The MPC starting population may be derived from any one or more tissuetypes set out in WO 01/04268 or WO 2004/085630, namely bone marrow,dental pulp cells, adipose tissue and skin, or perhaps more broadly fromadipose tissue, teeth, dental pulp, skin, liver, kidney, heart, retina,brain, hair follicles, intestine, lung, spleen, lymph node, thymus,pancreas, bone, ligament, bone marrow, tendon and skeletal muscle.

MEMPs can be distinguished from freshly harvested MPCs in that they arepositive for the marker STRO-1^(bri) and negative for the markeralkaline phosphatase (ALP). In a preferred embodiment of the presentinvention, at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%of the cells have the phenotype STRO-1^(bri), ALP⁻. In a furtherpreferred embodiment, the MEMPs are positive for one or more of themarkers Ki67, CD44 and/or CD49c/CD29, VLA-3, α3β1. In yet a furtherpreferred embodiment, the MEMPs do not exhibit TORT activity and/or arenegative for the marker CD18.

Once a suitable MPC population has been obtained, it may be cultured orexpanded by any suitable means to obtain MEMPs.

In a preferred embodiment of the invention, the MPCs are obtained from amaster cell bank derived from MPCs enriched from the bone marrow ofyoung healthy volunteers. The use of MPCs derived from such a source isparticularly advantageous for subjects who do not have an appropriatefamily member available who can serve as the MPC donor. Furthermore,other subjects, in particular those with acute leukemia are in fragileremissions prior to transplant and at high risk of relapsing during thelengthy time it takes to generate MPCs and then perform co-cultures. An“off-the-shelf” source offers major potential advantages over the familymember-derived. MPCs as the cells are available for immediate usewithout the need for lengthy processing or the possibility ofcontamination during culture. The development of master cell banksoffers a way to bypass disease-related decline in stem cell functionproviding the optimal source of MPCs for the cord blood co-cultures.

The Applicant has developed an off-the-shelf ex vivo expanded allogeneicMPC product for the treatment of chronic ischemic cardiovasculardisease, referred to as “Mesenchymal Precursor Cell” or Revascor™. Bonemarrow cells are harvested from the posterior iliac crest of healthyhuman donors. The mononuclear cells are immunoselected for stromalenrichment using the STRO-3 (TNSAP) monoclonal antibody (Simmons P J etal., (1991)), subsequently expanded, and cryopreserved to produce a cellbank. The expansion of immunoselected bone marrow mononuclear cellsconcentrated for mesenchymal precursors yields a product with definedpurity, expression of mesenchymal precursor specific markers, and potentbiological activity. Furthermore, work by the present Applicant andothers confirms the immunological tolerance of their allogeneic MPCs ina variety of preclinical and clinical allogeneic settings.

Since the Applicant's commercially available source of MPCs do notexpress HLA-II (DR), they are therefore non-immunogenic and provide anideal MPC source for use in the present invention.

Cord Blood

Exsanguination of the umbilical cord blood can be achieved by, forexample, but not by way of limitation, draining, gravity induced efflux,massaging, squeezing, pumping, etc. In a preferred embodiment,exsanguination of the umbilical cord is achieved by use of a syringethat may or may not include an anticoagulant.

Cord blood used in the methods of the present invention can be obtainedthrough a commercial source e.g. LifeBank USA (Cedar Knolls, N.J.),ViaCord (Boston Mass.), Cord Blood Registry (San Bruno, Calif.) andCryocell (Clearwater, Fla.).

Methods for harvesting cord blood cells are known in the art. Examplesof such methods are also described in the patent literature including,for example, U.S. Pat. No. 5,916,202 entitled “Umbilical cord bloodcollection” and U.S. Pat. No. 7,147,626 entitled “Cord blood andplacenta collection kit”.

In one embodiment, the cord blood is matched with the subject at 4, 5 or6/6 HLA class I (serological) and II (molecular) antigens. In anotherembodiment, at least two cord blood units are used per subject. The cordblood units may be cryogenically frozen prior to use or used straightaway when harvested from the umbilical cord.

Prior to culture, the cord blood cells may be enriched for CD34⁺progenitor cells, or they may be enriched for progenitor cells based onexpression of the marker CD133.

In one embodiment, the cord blood cells are unmanipulated prior to theiraddition to the MPCs.

In another embodiment, the cord blood cells are added to confluentmonolayers of pre-established MPCs or MEMPs.

In another embodiment, the cord blood units are co-cultured for around14 days in a suitable ex vivo expansion medium. The expansion mediumused for the co-culture may comprise fetal bovine serum, glutamine andsuitable growth factors.

In one embodiment of the invention, the number of expanded HPCs used totreat a subject is in the range of from ≧1.0×10⁷ TNC/kg to ≧4.0×10⁷TNC/kg.

Co-Culture Conditions

In one embodiment of the present invention, haematopoietic precursorcells, or unmanipulated cord blood cells, are added to an establishedadherent MPC cell culture. The MPCs may be cultured to confluence,replated and re-cultured to provide a feeder layer to which is added thecord blood cells for co-culturing.

One advantage of embodiments of present invention is that sufficientnumbers of confluent adherent allogeneic MPCs for co-culture can beobtained within about four days after initiating MPCs from a singlecryopreserved vial (containing ≧1.5×107 cells/ml at cryopreservation).Sufficient numbers of MPCs according to the invention is where enoughcells are present to reach >70% confluency in 12×T-150 cm² tissueculture flasks (per subject). This is considerably faster than prior artmethods of generating MPCs de novo from a bone marrow donor, whichtypically takes about four weeks.

In one embodiment of the invention, cord blood cells are co-culturedwith MPCs in an ex vivo expansion medium comprising fetal bovine serum,glutamine, G-CSF, SCF, FLT3-ligand and thrombopoietin.

The cells may be co-cultured for a period of about 14 days.

Administration of HPCs to the Subject

According to the methods of the present invention, the ex vivo expandedHPCs (which may or may not further include the co-cultured MPCs orMEMPs) are implanted into a subject having a haematological malignancy.In a preferred embodiment, the subject is a human.

Modes of administration of the cells include, but are not limited to,systemic intravenous injection. The cell preparation can be administeredby any convenient route, for example by infusion or bolus injection andcan be administered together with other biologically active agents.

In some embodiments, regimes for reducing implant rejection and/or graftvs. host disease of the HPCs may be practiced, particularly incircumstances where the allogeneic MPCs are also provided to thesubject. Such regimes are known in the art. See, for example Slavin S etal., J Clin Immunol. 2002 22:64. Typical GVHD prophylaxis agents includeantithymocyte globulin (ATG), mycophenalate mofetil (MMF), andtacrolimus.

It will be appreciated that the HPCs can be provided along with theculture medium supernatant or one or more factors derived fromco-cultured MPCs isolated from the culture medium, and administered in apharmaceutically acceptable carrier. Hence, cell populations of theinvention can be administered in a pharmaceutically acceptable carrieror diluent, such a sterile saline and aqueous buffer solutions. The useof such carriers and diluents in well known in the art.

In one example, the expanded HPCs are provided alone with MPCs or MEMPsof the co-culture. In another example, the HPCs are provided withoutMPCs or MEMPs. Methods for the separation of mesenchymal cells andhaemopoietic cells are well known in the art, and include, but arelimited to, affinity separation (according to markers present onmesenchymal cells and haemopoietic cells) by chromatography, batchseparation and/or flow cytometry (FACS).

MPCs, MEMPs or supernatant derived therefrom can be administered priorto, simultaneously with or after administration of the HPCs.

Haematopoietic Reconstitution

in one embodiment of the invention, haematopoietic reconstitution occursin the subject following administration of the expanded haematopoieticprecursor cells. For example, haematopoietic reconstitution may occur inthe subject within 30 days, more preferably within 25 days, morepreferably within 20 days, more preferably within 15 day and morepreferably within 10 days of administration of the expandedhaematopoietic precursor cells.

Haematopoietic reconstitution may determined by any one of a number ofsuitable measurements. For example, haematopoietic reconstitution istaken to have occurred once any one or more of the following haveoccurred: neutrophil engraftment, platelet engraftment, lymphoidengraftment, white blood cell engraftment, red blood cell engraftment,erythroid engraftment and/or megakaryocyte engraftment.

Neutrophil Engraftment

Neutrophil engraftment is defined as sustained absolute neutrophil count(ANC) which is greater than or equal to 0.5×10⁹/L for 3 consecutivedays.

Platelet Engraftment

Platelet engraftment is defined as the first of three consecutive dayswhen the unsupported platelet count was greater than 50×10⁹/L.

White Blood Cell Engraftment

White blood cell engraftment is defined as the first of threeconsecutive days an absolute polymorphonuclear cells (PMN) were greaterthan 50×10⁹/L.

Red Blood Cells (RBC) Engraftment

Red blood cells (RBC) engraftment may be recorded using measurement ofHbF and looking at F cells on blood films. For example, red blood cellengraftment may have occurred when the HbF is about 3.6% and the F cellsare about 7-8%.

The invention will now be described in more detail with reference to thefollowing non-limiting examples.

EXAMPLES Example 1 Comparison of “Off-the Shelf” Allogeneic MPCs withAutologous MSCs

Cord blood (CB) co-cultured with MSCs generated de novo from bone marrowwas compared with cord blood co-cultured with the Applicants“off-the-shelf” MPCs (Robinson et al., 2007).

MSCs are generated de novo from bone marrow as follows. Approximately80-100 ml of marrow is aspirated into sterile heparin-containingsyringes and taken to the MDACC Cell Therapy Laboratory for MSCgeneration. The bone marrow mononuclear cells are isolated usingficoll-hypaque and placed into two T175 flask with 50 ml per flask ofMSC expansion medium which includes alpha modified MEM (αMEM) containinggentamycin, glutamine (2 mM) and 20% (v/v) fetal bovine serum (FBS)(Hyclone).

The cells are cultured for 2-3 days in 37° C., 5% CO₂ at which time thenon-adherent cells will be removed; the remaining adherent cells will becontinually cultured until the cell confluence reaches 70% or higher(7-10 days), and then the cells will be trypsinized and replaced in sixT175 flasks with MSC expansion medium (50 ml of medium per flask). Thecells are cultured in 37° C., 5% CO₂ for an additional week. On day 14(+/−5 days) the six flasks of MSCs with 70% or higher confluence aresplit again into 12 flasks and cultured in MSC expansion medium for athird week as above. The MSC monolayers, which will be >70% confluent atthis time, are then ready for the CB expansion which should be initiatedon chemotherapy day −14. If the MSCs are ready before day −14, the MSCmonolayers are maintained by a weekly medium change with MSC culturemedium until ready for use.

Two frozen cord blood units were thawed, washed and co-cultured withadherent monolayers from each source (MSCs or MPCs) for 14 days usingthe presence of growth factors SCF, FLT3-ligand, G-CSF and TPO). Asshown in Table 1, allogeneic MPCs are better at expanding cord bloodCD34 progenitor cells than the allogeneic MSCs.

TABLE 1 Avg. TNC fold Avg. CD34+ fold Study “N” increase increaseAllogeneic MSC 6 13 14 cord expansion Pre-clinical 5 12.6 29 AllogeneicMPC cord expansion Clinical Allogeneic 9(TNCs); 16 39 MPC cord expansion11(CD34+)

Example 2 Time to Generation of Sufficient Numbers of “Off the Shelf”MPCs for Cord Blood Co-Culture

One vial containing frozen human mesenchymal precursor cells (Lot No.25126787, >1.5×10⁷ cells/ml) was thawed and 2.03×10⁶ cells recoveredinto 360 mls of alphaMEM medium supplemented with antibiotics(penicillin and streptomycin), glutamine and 10% (v/v) fetal bovineserum (FBS). The cell suspension was then distributed between 12×T-150cm² tissue culture flasks (approximately 1.7×10⁶ cells per T-150 cm²tissue culture flask). Cultures were monitored using inverted, phasecontrast microscopy. Four to five days after initiation of the culture,the MPCs were >70% confluent and could be used for cord bloodmononuclear cells (MNC) co-culture expansion. Accordingly, it is likelythat the cultured MPCs would be sufficiently confluent by four daysafter initiation of the culture. This indicates that a single vial ofoff-the-shelf MPCs are sufficient to generate sufficient numbers ofcells for cord blood co-culture after 3-5 days in culture. Thisrepresents a significant reduction from the approximately four weeks ofexpansion culture required to achieve sufficient numbers of confluentMPCs for co-culture from a bone marrow aspirate.

Example 3 Cord Blood Transplantation Protocols

3.1 Double Cord Blood Transplants

In an attempt to increase the number of CB cells infused followinghigh-dose or nonmyeloablative therapy, investigators have combined twounits of differing HLA types and infused them as allogeneichematopoietic support. These investigations support the principle thattransplantation of two immunologically distinct CB units is safe interms of crossed immunological rejection. Graft failure was notobserved, but the majority of patients did engraft with only a single CBunit. In the current trial we will use two CB units, one of which willbe expanded ex vivo in attempt to reduce the time to engraftment in CBtransplant recipients below the 20-30 days typically reported in thissetting.

3.2 Growth Factors for Ex-Vivo Expansion

The CB cells will be cultured with very low concentrations of the growthfactors described in this section (nanogram concentrations as opposed tothe microgram concentrations used systemically). Additionally, theexpanded cells will be washed extensively prior to infusion into thepatient. Thus, it is unlikely that they will produce any systemic sideeffects.

Suitable growth factors are described below.

1. Filgrastim [Granulocyte Colony Stimulating Factor (G-CSF)]

Therapeutic Classification: Recombinant Growth Factor

Mechanism of action: G-CSF is a human granulocyte-stimulating factorthat acts on hematopoietic cells to stimulate proliferation,differentiation, and some end-cell functional activity.

Storage and stability: G-CSF should be stored at 2-8° C. Prior toinjection, Filgrastim may be allowed to reach room temperature, however,any vials left at room temperature for greater than 24 hours should bediscarded. Vials should not be shaken. Vials should be inspected forsedimentation or discoloration prior to administration. If sedimentationor discoloration is observed, the vials should not be used.Route of administration: SC Injection-IV InfusionIncompatibility: No definite incompatibilities are known. However, drugsthat may potentiate the release of neutrophils should be used withcaution.Availability: Commercially available in single-dose, preservative-freevials containing 300 mcg (1 ml vial) and 480 mcg (1.6 ml vial) ofFilgrastim.Side effects: Mild to moderate hone pain is possible in patientsreceiving myelosuppressive therapy. General skin rash, alopecia, fevers,thrombocytopenia, osteoporosis, nausea, vomiting, diarrhea, mucositis,anorexia, inflammation of the blood vessels, and/or cardiac dysrhythmiacan occur. Splenomegaly may result at high doses of Filgrastim.2. Recombinant-metHuStem Cell Factor (SCF).Therapeutic Classification: Recombinant Growth FactorMechanism of action: SCF is a human granulocyte-stimulating factor thatacts on hematopoietic cells to stimulate proliferation, differentiation,and some end-cell functional activity.Pharmaceutical data: Recombinant methionyl human stem cell factor(r-metHuSCF) is a recombinant human protein produced in E. coli byrecombinant DNA technology. The 165-amino acid non-glycosylated proteincontains two intramolecular disulfide bonds, exists as a non-covalentlyassociated dimer with a molecular weight of 36,000, and differs from thenatural protein by the presence of a methionine moiety at the N-terminus(residue number [−1]) resulting from the expression in and in the factthat the recombinant protein is not glycosylated. Cells expressingr-metHuSCF are grown in culture under defined and controlled conditions.The cells are harvested yielding a paste from which the r-metHuSCF isextracted and purified via a series of proprietary processing andchromatographic steps. The resulting purified r-metHuSCF is formulatedin an aqueous buffer before undergoing sterile filtration and filling.Criteria for release of r-metHuSCF for use in the clinic are stringent.These include passing the USP rabbit pyrogen test, the limulus amebocyteassay, a sterility test, and the general safety test (Code of FederalRegulations, Title 21, Section 610.11). The nucleic acid content is nogreater than 1.7 pg/mg protein. The final product is a clear, colorless,sterile protein solution free of particulates; r-metHuSCF is not lessthan 95% pure. Biologic activity of purified preparations is assessedvia radioreceptor binding and proliferation assays.Storage and stability: Recombinant-metHuSCF must be stored at 2-8° C.Stability of r-metHuSCF at concentrations of 1.5 mg/ml has beendemonstrated for 12 months when stored under these conditions. Stabilitytesting is ongoing. Exposure of the material to excessive temperaturesabove or below this range is to be avoided. Do not allow r-metHuSCF tofreeze, and do not use if contents freeze in transit or in storage.SIDE EFFECTS: Not known but injection site complication such asurticaria and erythema3. Thrombopoietin (TPO)Therapeutic Classification: Recombinant Growth FactorMECHANISM OF ACTION: TPO is a human granulocyte-stimulating factor thatacts on hematopoietic cells and in particular megarkyocyte progenitorsto stimulate proliferation, differentiation, and some end-cellfunctional activity.Storage and stability: TPO should be stored at 2-8° C. Vials should notbe shaken. Vials should be inspected for sedimentation or discolorationprior to administration. If sedimentation or discoloration is observed,the vials should not be used.Known side effects: thrombocytosis, deep-vein thrombosis, pulmonaryembolism, thrombophlebitis.4. FLT3-Ligand (FLT3)Therapeutic Classification: Recombinant Growth FactorMechanism of action: FLT3 is a human granulocyte-stimulating factor thatacts on hematopoietic cells.Storage and stability: FLT3 should be stored at 2-8° C. Vials should beinspected for sedimentation or discoloration prior to administration. Ifsedimentation or discoloration is observed, the vials should not beused.Known side effects: thrombocytosis, deep-vein thrombosis, pulmonaryembolism, thrombophlebitis.3.3 Administration of Off-the-Shelf MPCs.

Angioblast Revascor™ MPCs are used for the CB co-cultures. A singlefrozen vial of Angioblast Revascor™ MPC will supply sufficient cells toseed three (3) to four (4) T-300 cm2 culture flasks. After recovery fromthawing and after a period of 2-3 days of culture in αMEM mediumsupplemented with ten (10) percent fetal bovine serum (MPC culturemedium), adherent cells will be collected by trypsinization and thewhole transferred into twelve (12) T-150 cm2 culture flasks. Cells willbe cultured in MPC culture medium in the twelve (12) T-150 cm2 cultureflasks for an additional five (5) to six (6) days until growthsufficient to achieve greater than 70% confluence over the availableculture surface is achieved. At this point the T-150 cm2 culture flaskswill be released for use in the cord blood co-culture expansionprotocol. Culture flasks generated in this manner can be maintainedat >70% confluence until required by a weekly medium change. Ten (10) ofthe twelve (12) T-150 cm2 culture flask generated and containing MPCat >70% confluence will receive ten (10) percent of a thawed, washedcord blood unit. Co-culture will be performed in culture mediumsupplemented with fetal bovine serum and containing 100 ng/ml each ofSCF, FLT3-L, TPO and G-CSF as described above for the familymember-derived MSCs.

Patients will be admitted to the hospital on day −9 for hydration andreceive the designated preparative regimen on days −8 through −2. On day0, the unmanipulated CB unit will be infused followed by the expanded CBunit. On day 0 (culture day 14), the cells from both cultures will beharvested and washed for infusion.

Example 4 Clinical Trial Results

Fourteen patients were transplanted with MPC induced expanded cord bloodin an ongoing trial. The average age of the patient was 40 years.Current results of the transplants are shown in Table 2 below.

The median time to neutrophil engraftment was 17 days (compared tohistoric controls of 34 days). The median time to platelet engraftmentwas 38 days (compared to historic controls of 128 days). No patients hadGrade III/IV GVHD (compared to historic controls of 40%).

Table 3 shows that current MPC induced expanded cord bloodtransplantation results are superior to alternative cord blood expansionstrategies.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the scope of theinvention as broadly described. The present embodiments are, therefore,to be considered in all respects as illustrative and not restrictive.

TABLE 2 Date of Time to neutrophil Platelet Patient transplantengraftment engraftment GVHD 1 9 Dec. 2008 Day 12 (21 Dec. 2008) Day 57(04 Feb. 2009) BX positive GVDH GI Duodenum 2 10 Feb. 2009 Day 22 (4Mar. 2009) Day 18 (28 Feb. 2009) Negative 3 13 Feb. 2009 Day 15 (28 Feb.2009) Day 37 (22 Mar. 2009) Negative 4 12 May 2009 Day 13 (25 May 2009)Day 29 (10 Jun. 2009) Negative 5 19 May 2009 Day 19 (7 Jun. 2009) Day 45(3 Jul. 2009) BX positive GVDH GI Duodenum, stomach, colorectal 6 4 Jun.2009 Day 25 (29 Jun. 2009) Day 39 (13 Jul. 2009) Negative 7 15 Jun. 2009N/A N/A Negative 8 23 Jun. 2009 Day 24 (17 Jul. 2009) N/A 9 Off studyOff study Off study Off study 10 6 Jul. 2009 Day 9 (15 Jul. 2009) N/A 113 Aug. 2009 N/A N/A 12 21 Jul. 2009 N/A N/A 13 23 Jul. 2009 N/A N/A 14 7Aug. 2009 N/A N/A

TABLE 3 Rate of Median Median GVHD TNC CD34 time to time to as GradeExpansion Expansion neutrophil platelet 2 or Product “N” fold Foldengraftment engraftment Greater 2 Angioblast 14 16 39 17 38  0%Allogeneic MPC *Aastrom 28 2.4 0.5 22 71 36% Biosciences Replicall ™**Viacell 10 219 24 54 40% Selected Amplification ™ ***Gamida 10 6 30 4844% G2, StemEx ™ 50% chronic GVHD post day 100 ****Historic 97 adultsN/A N/A 28 90 46% Control (New (560 (Adults York Blood patients andCenter) including Pediatric pediatric) combined rate) *Jaroscak et al.,2003 **Chan et al., 2006 ***de Lima et al., 2008 ****Rubinstein et al.,1998

REFERENCES

-   Barker, J. N., S. M. Davies, T. DeFor, N. K. Ramsay, D. J. Weisdorf,    and J. E. Wagner, Survival after transplantation of unrelated donor    umbilical cord blood is comparable to that of human leukocyte    antigen-matched unrelated donor bone marrow: results of a    matched-pair analysis. Blood, 2001. 97(10): p. 2957-61.-   Chan, S. L et al., Enhanced in vivo homing of uncultured and    selectively amplified cord blood CD34+ cells by co-transplantation    with cord-blood derived unrestricted somatic stem cells. Stem    Cells, 2006. 25:529-536.-   De Lima, M. et al. Transplanatation of ex vivo expanded cord blood    cells using the copper chelator tetraethylenepentamine: a phase I/II    clinical trial. Bone Marrow Transplantation, 2008. 1-8.-   Gluckman, E. V. Rocha, A. Boyer-Chammard, F. Locatelli, W.    Arcese, R. Pasquini, J. Ortega, G. Souillet, E. Ferreira, J. P.    Laporte, M. Fernandez, and C. Chastang, Outcome of cord-blood    transplantation from related and unrelated donors. Eurocord    Transplant Group and the European Blood and Marrow Transplantation    Group. N. Engl. J. Med., 1997. 337(6): p. 373-81.-   Jaroscak, J. et al., Augmentation of umbilical cord blood    transplantation with ex-vivo expanded UCB cells: results of phase I    trial using Aastrom Replicell system. Blood, 2003. 101:5061-5067.-   Kernan, N. A., G. Bartsch, R. C. Ash, P. G. Beatty, R. Champlin, A.    Filipovich, J. Gajewski, J. A. Hansen, J. Henslee-Downey, and J.    McCullough, Analysis of 462 transplantations from unrelated donors    facilitated by the National Marrow Donor Program. N. Engl. J.    Med., 1993. 328(9): p. 593-602.-   Kurtzberg, J., M. Laughlin, M. L. Graham, C. Smith, J. F.    Olson, E. C. Halperin, G. Ciocci, C. Carrier, C. E. Stevens, and P.    Rubinstein, Placental blood as a source of hematopoietic stem cells    for transplantation into unrelated recipients. N. Engl. J.    Med., 1996. 335(3): p. 157-66.-   Laughlin, M. J., J. Barker, B. Bambach, O. N. Koc, D. A.    Rizzieri, J. E. Wagner, S. L. Gerson, H. M. Lazarus, M. Cairo, C. E.    Stevens, P. Rubinstein, and J. Kurtzberg, Hematopoietic engraftment    and survival in adult recipients of umbilical-cord blood from    unrelated donors. N. Engl. J. Med., 2001. 344(24): p. 1815-22.-   Rizzieri, D. A., G. D. Long, J. J. Vredenburgh, C. Gasparetto, A.    Morris, D. Niedzwiecki, M. Lassiter, J. Loftis, P. Davis, C.    McDonald, T. Stenzel, B. Waters-Pick, J. Kurtzberg, and N. J. Chao,    Durable Engraftment of Mismatched Unrelated Cord Blood Following a    Non-Myeloablative Prepative Regimen for Adults. Blood, 2001. 98(11    (1)): p. 185a.-   Robinson, S, N. et al., Efficacy of “Off-the-Shelf” commercially    available, third-party mesenchymal stem cells (MSC) in ex vivo cord    blood (CB) co-culture expansion. Blood (ASH Annual Meeting    Abstracts) 2007 110: Abstract 4106.-   Rubinstein, P., C. Carrier, A. Scaradavou, J. Kurtzberg, J.    Adamson, A. R. Migliaccio, R. L. Berkowitz, M. Cabbad,    Dobrila, P. E. Taylor, R. E. Rosenfield, and C. E. Stevens, Outcomes    among 562 recipients of placental-blood transplants from unrelated    donors. N. Engl. J. Med., 1998. 339(22): p. 1565-77.

The invention claimed is:
 1. A method of transplanting haematopoieticprecursor cells into a subject in need thereof, the method comprising:culturing haematopoietic precursor cells in the presence of a populationof cells enriched for STRO-1^(bright) cells or supernatant or progenyderived therefrom, wherein such STRO-1^(bright) cells are mesenchymalprecursor cells (MPC) which comprise mesenchymal precursor cells capableof giving rise to colony forming unit-fibroblasts (CFU-F), so as toexpand the haematopoietic precursor cells; and administering theexpanded haematopoietic precursor cells to a subject, whereinhaematopoietic reconstitution occurs in the subject within 10 to 30 daysof administration of the expanded haematopoietic precursor cells.
 2. Themethod of claim 1, wherein the population of cells enriched forSTRO-1^(bright) cells are allogeneic cells.
 3. The method of claim 1wherein the expanded haematopoietic precursor cells comprise CFU-GMcells.
 4. The method of claim 1 wherein haematopoietic reconstitutionoccurs in the absence of an adverse immune response.
 5. The method ofclaim 1 wherein haematopoietic reconstitution is determined byneutrophil engraftment, platelet engraftment, lymphoid engraftment,erythroid engraftment and/or megakaryocyte engraftment.
 6. The method ofclaim 1 which further comprises administering to the subject a factorwhich enhances differentiation of the haematopoietic precursor cells toa specific haematopoietic lineage cell.
 7. The method of claim 6 whereinthe haematopoietic lineage cell is a B-cell, T-cell, dendritic cell,monocyte, neutrophil, macrophage, natural killer cell, granulocyte,erythrocyte, eosinophil, megakaryocyte, platelet, bone marrow, splenic,dermal, or stromal cell.
 8. The method of claim 6 wherein the factorwhich enhances differentiation is stem cell factor (SCF), GM-SCF, M-CSF,G-CSF, MGDF, EPO, FLT3-ligand, IL-1, IL-2, IL-3, IL-4, IL-6, IL-7,IL-11, TNFα, or thrombopoietin.
 9. The method claim 1 whereinadministering the expanded haematopoietic precursor cells reduces therisk of host versus graft disease when compared to administration ofhaematopoietic precursor cells that have not been subject to ex vivoexpansion.
 10. The method of claim 1 wherein the subject has ahaematologic disorder.
 11. The method of claim 10 wherein thehaematologic disorder is a disorder of platelet number and/or function.12. The method of claim 11 wherein the haematologic disorder isthrombocytopenia, idiopathic thrombocytopenic purpure (ITP), or adisorder related to viral infection, drug abuse or malignancy.
 13. Themethod of claim 10 wherein the haematologic disorder is a disorder oferythrocyte number and/or function.
 14. The method of claim 13 whereinthe haematologic disorder is an anaemia.
 15. The method of claim 14wherein the anaemia is aplastic anaemia, autoimmune haemolytic anaemia,blood loss anaemia, Cooley's anaemia, Diamond-Blackfan anaemia, Fanconianaemia, folate (folic acid) deficiency anaemia, haemolytic anaemia,iron-deficiency anaemia, pernicious anaemia, sickle cell anaemia,thalassaemia or Polycythemia Vera.
 16. The method of claim 10 whereinthe haematologic disorder is a disorder of lymphocyte number and/orfunction.
 17. The method of claim 16 wherein the disorder is due to aT-cell or B-cell deficiency.
 18. The method of claim 16 wherein thedisorder is AIDS, a leukemia, a lymphoma, Hodgkins lumphoma, a chronicinfection such as military tuberculosis, a viral infection, rheumatoidarthritis, systemic lupus erythaematosus, or a hereditary disorder suchas agammaglobulinemia, DiGeorge anomaly, Wiskott-Aldrich syndrome, orataxia-telangiectasia.
 19. The method of claim 10 wherein thehaematologic disorder is a disorder of multilineage bone marrow failure.20. The method of claim 19 wherein the haematologic disorder is a resultof radiotherapy or chemotherapy.
 21. The method of claim 19 wherein thehaematologic disorder is a result of malignant replacement.
 22. Themethod of claim 21 wherein the haematologic disorder is myelofibrosis,acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), acutelymphoblastic leukemia (ALL), chromic myelogenous leukemia (CML),chronic lymphocytic leukemia (CLL), Non-Hodgkin's lymphoma (NHL),Hodgkin's Disease (HD), multiple myeloma, or a secondary malignancydisseminated to bone.